In nature, fertilisation occurs by sperm cells being deposited into the female of warm-blooded animal species (including humans) and then binding to and fusing with an oocyte. This fertilised oocyte then divides to form an embryo. Over the last several decades, the use of assisted reproduction techniques has allowed scientists and clinicians to intervene in these events to treat poor fertility in some individuals or to store sperm, oocytes or embryos for use at other locations or times. In vitro fertilisation (IVF) involves collection of eggs e.g. by laparoscopy, followed by mixing eggs and sperm and culture of the embryos before transferring them to the uterine cavity of the female to continue their development. A recent innovation has been the introduction of the technique of intracytoplasmic sperm injection (ICSI), in which sperm are microinjected into the egg cytoplasm.
At each step of the way, in vitro intervention decreases the normal survival and function of sperm, oocytes, and embryos. Much research has been dedicated towards improving these procedures; however, overall success remains limited. For example, less than 20% of IVF attempts result in the birth of a child. Oocytes and embryos also show significantly disrupted function after culture. Thus, in spite of several decades of work, much room remains for improvement in the field of assisted reproduction technologies and especially in gamete and embryo handling, culture, and storage.
There is a particular need for improved culture conditions to support mammalian oocytes and embryos. A high percentage of embryos that are fertilised or transferred in vitro cease development prematurely. The consequences are felt at both the economic and the human levels. Many programs of fertility treatments for humans involve the in vitro fertilisation or transfer of oocytes or embryos. The success rates of human fertility treatments are not high. The low success rates impose substantial economic and emotional costs. Even incremental improvements in the success rate can be of substantial benefit. One of the many causes of the low overall success rate is the frequent failure of embryos to grow and develop properly in vitro. Improved culturing conditions to better support embryo growth can not only enhance the success rate of fertility treatments, but ironically can also reduce the rate of multiple pregnancies resulting from the treatments. Because the overall success rate of current methods is low, practitioners often implant multiple embryos to increase the likelihood of pregnancy. Implanting multiple embryos increases the likelihood of multiple pregnancies as well. If each individual embryo were more likely to survive, then the perceived need to implant multiple embryos simultaneously would decline, and the rate of multiple pregnancies would decrease.
Prior art describes different culture conditions. In U.S. Pat. No. 6,140,121 nitric oxide adversely affects survival and development of cells such as oocytes and embryos in vitro, particularly in a co-culture system. The addition of a nitric oxide inhibitor such as hemoglobin to such systems eliminates this toxic effect, and promotes mammalian oocytes, embryos, or other cells in vitro.
U.S. application 20010028878 describes a method for in vitro maturation of a human oocyte by culturing an immature human oocyte in a cell culture medium for 10-30 hours. The maturation end point is metaphase II.
U.S. Pat. No. 6,140,121 describe methods and compositions to improve germ cell and embryo survival and function. Sperm, oocyte, and embryo survival and function is improved in vivo or in vitro by the use of a polysaccharide containing arabinose, galactose and/or hexuronic acid. In particular, a nonspermicidal lubricant containing such a polysaccharide (e.g., gum arabic, pectin, or galacturonic acid) increases the fertilisation potential of the sperm during coitus, artificial insemination or sperm collection.
Similarly, a freezing medium containing a polysaccharide containing arabinose, galactose and/or hexuronic acid enhances sperm, oocyte, or embryo viability.
U.S. Pat. No. 6,110,741 describe Gonadotrophin releasor hormone-containing composition for embryo culture and method for in vitro fertilisation. In vitro incubation of primate embryos in the presence of added exogenous gonadotrophin releasor hormone (GnRH), results in enhanced chorionic gonadotrophin production associated with increased survival and attachment of the embryos. Treatment of in vitro fertilised embryos with GnRH can be used to improve implantation. Agonists of GnRH reduce attachment competence of embryos and are thereby useful as post-fertilisation contraceptives.
U.S. Pat. No. 6,050,935 describes an intravaginal fertilisation and culture container including a main chamber and a micro chamber.
U.S. Pat. No. 5,882,928 describe in vitro maturation and fertilisation of mammalian oocytes. An in vitro fertilisation method comprising culturing mammalian immature oocytes obtained from a mammalian ovary very early in the mammal's menstrual cycle in an oocyte maturation inhibitor-containing culture, removing the inhibitor and maturing and fertilising the oocytes to produce embryos for subsequent uterine-implantation. Preferably, the inhibitor is dbcAMP.
U.S. Pat. No. 5,837,543 describes a Human embryo co-culture system wherein an oocyte from a female is contacted with a monolayer of cultured human tubal epithelial cells; inseminating the oocyte; and transferring an embryo back to said female.
U.S. Pat. No. 5,730,777 describes an apparatus for performing operations under a gaseous atmosphere in a closed chamber comprising performing the operation in a housing comprising i) first chamber walls defining a first chamber containing a gaseous atmosphere and ii) second chamber walls defining a second chamber substantially enclosing the first chamber, the second chamber containing a gaseous atmosphere between the first and second chamber walls.
U.S. Pat. No. 4,892,830 describes an environmentally controlled incubator in which the enclosure of the incubator chamber strongly attenuates the transmission of light having wavelengths below about 500 nm for protecting biological materials within the chamber from toxic effects of short wavelength light. The incubator also includes sensors for determining the oxygen and carbon dioxide concentration within the chamber and means for adding carbon dioxide, nitrogen or oxygen to the ambient gas within the incubator ion order to maintain the desired levels of carbon dioxide and oxygen. It has been found that the normal oxygen concentration in air (21%) is toxic to mouse zygotes and pre-embryos. If the oxygen level is reduced to 5-10% however, this inhibition is not observed and the embryos continue to develop.
WO9830676 describes a method and incubator for cells and tissues, in particular sensitive cells and tissues, such as oocytes, fertilised oocytes and pre-implantation embryos, which require highly stable physical and chemical environment for in vitro development, are cultured in closed containers submerged or immersed in thermostatically controlled liquid baths, the containers being provided with an appropriate inner atmosphere containing, e.g. carbon dioxide, oxygen and humidity in appropriate levels
WO9967364 describes a medium for the propagation of early stage embryos to blastocyst stage. The medium contains an effective amount of human GM-CSF to increase the percentage of pre-blastocyst embryos which develop to transfer ready blastocysts. Also it describes a method of growing early stage human embryos to transfer ready blastocysts. The method includes the step of incubating the embryos in vitro in a culture medium containing an effective amount of human GM-CSF for a time and under conditions to increase the proportion of transfer ready blastocysts. An IVF program that includes the method of growing early stage human embryos to transfer ready blastocysts is also described.
WO0032140 describes the following: Instead of immersing human reproductive cells in a single culture medium throughout the various procedures used in IVF, a process is provided by which the reproductive cells may be moved through a sequence of distinct culture media as the various IVF procedures are carried out.